Functional Annotation of the Preimplantation Transcriptome. The primary goal of this proposal is to elucidate the requirement of a large set of genes expressed during mammalian preimplantation development. After RNAi mediated loss of each gene function, we will assess morphological development, lineage specification, epigenetic regulation of genome imprinting and embryonic stem cell derivation potential. This work will extend a pilot RNAi screen that we have performed (~500 genes) in order to establish conditions for this transcriptome-wide effort. Currently there exist sufficient data sets such that we will not need to determine which genes are expressed ourselves - but can use the work of other groups in order to launch directly towards our goal of functional annotation of the preimplantation transcriptome. Our system capitalizes on the dynamic genome reprogramming that occurs during the first few days post-fertilization. Our approach uses mouse embryos as an in vivo model system and monitors early developmental events and genomic imprinting. Our approach provides a robust and efficient method towards identification of novel epigenetic regulatory mechanisms as well as functional requirements during development and we are uniquely poised to carry out the proposed work. In addition to the bolus of basic scientific knowledge our data will provide, our proposal is highly relevant for understanding the evidence that even short-term exposure to in vitro culture during preimplantation can have dire epigenetic consequences. Perhaps even more relevant are numerous examples of epigenetic defects in embryos and placentas of individual mammals generated through the use of artificial reproductive technologies. Understanding the basic molecular machinery that regulates epigenetic events during preimplantation is of critical importance if we are to improve current practice as well as to design therapeutic approaches for the many human disease states with epigenetic mis-regulation of gene expression.